Abstract

Treatment of intervertebral disc degeneration (IDD) seeks to prevent senescence and death of nucleus pulposus (NP) cells. Previous studies have shown that sirt6 exerts potent anti-senescent and anti-apoptotic effects in models of age-related degenerative disease. However, it is not known whether sirt6 protects against IDD. Here, we explored whether sirt6 influenced IDD. The sirt6 level was reduced in senescent human NP cells. Sirt6 overexpression protected against apoptosis and both replicative and stress-induced premature senescence. Sirt6 also activated NP cell autophagy both in vivo and in vitro. 3-methyladenine (3-MA) and chloroquine (CQ)-mediated inhibition of autophagy partially reversed the anti-senescent and anti-apoptotic effects of sirt6, which regulated the expression of degeneration-associated proteins. In vivo, sirt6 overexpression attenuated IDD. Together, the data showed that sirt6 attenuated cell senescence, and reduced apoptosis, by triggering autophagy that ultimately ameliorated IDD. Thus, sirt6 may be a novel therapeutic target for IDD treatment.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1. Sirt6 level declines in senescent…

Fig. 1. Sirt6 level declines in senescent NP cells both in vivo and in vitro

Fig. 1. Sirt6 level declines in senescent NP cells both in vivo and in vitro

a Immunofluorescence of sirt6 in aging (16 months) group and young (3 months) group (scale bar: 200 μm). b Immunofluorescence of sirt6 in NP cells that were isolated from aging and young rats (scale bar: 50 μm). c, d Representative western blots and quantification data of sirt6 in NP cells of each group. e SA-β-gal staining assay was performed in rat NP cells as treated above (scale bar: 50 μm). f–h Representative western blots and quantification data of p16 and sirt6 in NP cells of each group. Columns represent mean ± SD. Significant differences between the treatment and control groups are indicated as *P < 0.05, **P < 0.01, ***P < 0.001, n = 5

We passaged NP cells to the 15th generations to detect sirt6 and p16. a SA-β-gal staining assay was performed in NP cells of each group (scale bar: 50 μm). b–d Representative western blots and quantification data of p16 and sirt6 in human NP cells of each group; columns represent mean ± SD. Significant differences between the treatment and sham groups are indicated as **P < 0.01, ***P < 0.001, n = 5

a, b TUNEL assay was performed to assess the apoptosis in NP cells of each group as treated above (scale bar: 50 μm). c–f Representative western blots and quantification data of cleaved caspase3, Bax, Bcl-2 in NP cells of each group as treated above. g Caspase3 activity in NP cells of each group. Columns represent mean ± SD. Significant differences between the treatment and control groups are indicated as *P < 0.05, n = 5

a SA-β-gal staining assay was performed in NP cells of each group as treated above (scale bar: 50 μm). b–e Representative western blots and quantification data of p-p53, p53, p21, and p16 in NP cells of each group as treated above; columns represent mean ± SD. Significant differences between the treatment and control groups are indicated as *P < 0.05, **P < 0.01, ***P < 0.001, n = 5

a, b T2-weighted MRI and relative Pfirrmann MRI grade scores of a rat tail with a needle-punctured disc at 8 weeks in each group (white arrows); columns represent mean ± SD. Significant differences between the treatment and control groups are indicated as *P < 0.05, ***P < 0.001, n = 5. c HE staining of each group. d, e Immunohistochemical staining of cleaved-caspase3 and LC3-II expression in the disc samples of each group (scale bar: 200 μm)